Ruxolitinib-based senomorphic therapy mitigates cardiomyocyte senescence in septic cardiomyopathy by inhibiting the JAK2/STAT3 signaling pathway

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  Figure 1. Cellular senescence is induced in SCM: evidence from human, animal, and cell models. (A,B) Representative Western blot bands and quantitative analysis of P16, P21, P53, and γ-H2AX in NRCMs between the control and LPS-stimulation groups. N = 4. (C) The mRNA levels of P16, P21 and P53 in NRCMs between the control and LPS-stimulation groups were detected using qRT-PCR. N = 4. (D,E) Representative fluorescence images and quantitative analysis of P16 with DAPI in NRCMs between the control and LPS-stimulation groups. Scale bar = 20 μm, N = 3. (F,G) Representative images and quantitative analysis of SA-β-gal staining in NRCMs. Scale bar = 20 μm, N = 3. (H) The mRNA levels of SASP-related genes were detected using qRT-PCR post LPS-stimulation in NRCMs compared to the control group, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, CCL5 and GDF15. N = 3 - 4. (I,J) Representative Western blot bands and quantitative analysis of P16, P21, P53 and γ-H2AX in LPS-induced mice models compared with the Sham group. N = 6. (K) The mRNA levels of P16, P21, P53 in LPS-induced mice models compared with the Sham group were detected using qRT-PCR. N = 6. (L,M) Representative fluorescence images…

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  Figure 2. RNA-seq and bioinformatics analysis reveal the potential effects of Rux treatment on mouse hearts. (A) PCA identified the mice as two groups, namely the SCM group and the Rux group. N = 4 in each group. (B) DEGs between the SCM group and the Rux treatment group were shown in the volcano map. (C) KEGG analysis identified changes in the cardiac pathways of mice with SCM following Rux therapy. (D) The heat map indicated that senescence markers, specifically P53 and P21, were downregulated in mice hearts with SCM after Rux treatment. N = 4 in each group. (E) The relative levels of SASP-related genes were displayed in the heatmap between the SCM and the Rux groups, including CXCL1, CXCL5, MMP3, IL-1α, TGFβ2, CXCL14, IL-6, CXCL10, GDL15, and IL-1β. N = 4 in each group. (F) GSEA analysis demonstrated that the JAK-STAT pathway was up-regulated in SCM-induced mouse hearts and down-regulated after treatment with Rux. (G) GO analysis identified changes in the cardiac biological processes of SCM mice following Rux therapy. (H) The heatmap demonstrated that the relative levels of MRC complex-related genes were upregulated post Rux treatment in SCM-induced mice hearts. N = 4 in each group. (I)…

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  Figure 3. Senomorphic therapy with Rux exerts an anti-SASP effect and attenuates LPS-induced cellular senescence in NRCMs. (A) Schematic diagram of in vitro experiments in NRCMs. (B) The mRNA levels of SASP-related genes were detected using qRT-PCR in NRCMs treated with Rux or not post LPS-stimulation, including IL-1β, IL-6, TNF-α, CXCL1, CXCL3, CXCL10, CCL2, CCL5 and GDF15. N = 4. (C,D) Representative Western blot bands and quantitative analysis of P16 and P21 in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (E) The mRNA levels of P16, P21, and P53 were detected using qRT-PCR in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (F-I) Representative fluorescence images and quantitative analysis of P16 and P21 with DAPI in NRCMs treated with Rux or not post LPS stimulation. Scale bar = 20 μm, N = 3. (J,K) Representative images and quantitative analysis of SA-β-gal staining in NRCMs treated with Rux or not post LPS stimulation. Scale bar = 20 μm, N = 3. (L,M) Representative Western blot bands and quantitative analysis of γ-H2AX in NRCMs treated with Rux or not post LPS stimulation. N = 4. Data are presented…

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  Figure 4. Senomorphic therapy with Rux exerts an anti-SASP effect and attenuates cellular senescence in mouse models with SCM. (A) The mRNA levels of SASP-related genes were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS-injection compared with the Sham group, including CXCL1, CXCL3, CCL5, TGFβ2 and GDF15. N = 4. (B,C) Representative Western blot bands and quantitative analysis of P16, P21, and P53 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (D) The mRNA levels of P16, P21, and P53 were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (E,F) Representative fluorescence images and quantitative analysis of P21 with cTnI and DAPI in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (G,H) Representative Western blot bands and quantitative analysis of γ-H2AX in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4.…

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  Figure 5. Rux treatment alleviates cardiac dysfunction and mitochondrial-mediated apoptosis in mice with SCM. (A) Schematic diagram of in vivo experiments in LPS-induced mouse models. (B) Schematic diagram of in vivo experiments in CLP-induced mouse models. (C,D) Cardiac function indices were measured by echocardiography in mice treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 5. (E,F) Cardiac function indices were measured by echocardiography in mice treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP surgery compared with the Sham group. N = 5. (G,H) Representative images and quantitative analysis of TUNEL assays in in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (I,J) Representative immunohistochemistry images and quantitative analysis of Cyto-c expression levels in in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (K,L) Representative Western blot bands and quantitative analysis of cyto-c and Bax in in NRCMs treated with Rux or not post…

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  Figure 6. Senomorphic therapy with Rux alleviates oxidative stress and mitochondrial dysfunction in SCM. (A) The mRNA levels of Sirt3 were detected using qRT-PCR in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 3. (B,C) Representative Western blot bands and quantitative analysis of Ac-SOD2, SIRT3 and SOD2 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (D) Relative SOD2 activity in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 3. (E) Relative MDA levels in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 3. (F-I) Representative fluorescence images and quantitative analysis of ROS and MitoSox in NRCMs treated with Rux or not post LPS stimulation. Scale bar = 20 μm, N =3. (J) Representative TEM images of mitochondria in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. Scale bar = 0.5 μm. (K,L) Representative fluorescence images and quantitative…

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  Figure 7. Senomorphic therapy with Rux restores mitochondrial dynamics and alleviated endoplasmic reticulum stress in SCM. (A,B) Representative Western blot bands and quantitative analysis of p-Drp1, Fis1, Mfn2 and Opa1 in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (C,D) Representative Western blot bands and quantitative analysis of p-Drp1, Mfn2 and Opa1 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (E-H) Representative fluorescence images and quantitative analysis of Opa1 and p-Drp1 with TOMM20 and DAPI in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP surgery compared with the Sham group. Scale bar = 20 μm, N = 3. (I,J) Representative Western blot bands and quantitative analysis of GRP78, ATF4 and CHOP in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (K,L) Representative Western blot bands and quantitative analysis of GRP78, ATF4 and CHOP in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (M,N) Representative…

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  Figure 8. Senomorphic therapy with Rux regulates mitochondrial dynamics and mitochondrial-mediated apoptosis by inhibiting the JAK2-STAT3 signaling pathway. (A,B) Representative Western blot bands and quantitative analysis of p-JAK2 and p-STAT3 in NRCMs treated with Rux or not post LPS stimulation compared with the control group. N = 4. (C,D) Representative Western blot bands and quantitative analysis of p-JAK2 and p-STAT3 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post LPS injection compared with the Sham group. N = 4. (E,F) Representative Western blot bands and quantitative analysis of p-JAK2 and p-STAT3 in mouse hearts treated with 30 mg/kg and 75 mg/kg of Rux or saline post CLP surgery compared with the Sham group. N = 4. (G,H) Representative Western blot bands and quantitative analysis of p-JAK2 and p-STAT3 after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (I,J) Representative Western blot bands and quantitative analysis of p-Drp1, Fis1, Mfn2 and Opa1 after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (K,L) Representative fluorescence images and quantitative analysis of ROS and MitoSox after reactivating the JAK2-STAT3…

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  Figure 9. Inhibition of the JAK2-STAT3 pathway is critical for the protective effects of Rux in alleviating cellular senescence and SASP in SCM. (A) The mRNA levels of SASP-related genes were detected using qRT-PCR after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs, including IL-1β, IL-6, TNF-α, CXCL3, CXCL10, CCL2 and GDF15. N = 4. (B,C) The mRNA levels of P16 and P21 were detected using qRT-PCR after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (D,E) Representative Western blot bands and quantitative analysis of P16 and P21 after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 4. (F,G) Representative fluorescence images and quantitative analysis of γ-H2AX after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. N = 3. (H,I) Representative images and quantitative analysis of SA-β-gal staining after reactivating the JAK2-STAT3 pathway using r-IL6 in NRCMs. Scale bar = 20 μm, N = 3. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. LPS, Lipopolysaccharide; Rux, ruxolitinib; Con, control; r-IL6, recombinant IL-6; SA-ß-gal, senescence-associated β-galactosidase; NRCMs, neonatal rat cardiomyocytes.

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  Figure 10. Low-dose Rux and ABT263 demonstrated comparable efficacy in ameliorating SCM. (A,B) Cardiac function indices were measured by echocardiography in mice treated with Rux or ABT263 post LPS injection compared with the Sham group. N = 5. (C,D) Representative Western blot bands and quantitative analysis of P16 and P21 in LPS-induced NRCMs treated with ABT263 or Rux. N = 4. (E) The mRNA levels of P16 and P21 were detected using qRT-PCR in NRCMs treated with ABT263 or Rux. N = 5. (F) Representative fluorescence images and quantitative analysis of P16 with cTnI and DAPI in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group. Scale bar = 20 μm, N = 3. (G) The mRNA levels of SASP-related genes were detected using qRT-PCR in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group, including CXCL1, CXCL3, CXCL10, CCL2, IL-6, and GDF15. N = 5. (H) Representative images of SA-β-gal staining in mouse hearts treated with Rux or ABT263 post LPS injection compared with the Sham group. Scale bar = 50 μm. Data are presented as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Lipopolysaccharide; Rux, ruxolitinib; SA-ß-gal,…